Báo cáo khoa học: Recent contributions of in vitro models to our understanding of hepatitis C virus life cycle pdf

REVIEW ARTICLE Recent contributions of in vitro models to our understanding of hepatitis C virus life cycle ´ ` Morgane Regeard, Charlotte Lepere, Maud Trotard, Philippe Gripon and Jacques Le Seyec ˆ INSERM, U522, IFR 140, Hopital de Pontchaillou, Rennes, France Keywords assembly; hepatitis C virus; in vitro models; infection; replication Correspondence ˆ J Le Seyec, INSERM U522, Hopital Pontchaillou, Avenue Henri Le Guilloux, Rennes, F-35033, France Fax: +33 99 54 01 37 Tel: +33 99 54 74 07 E-mail: jacques.leseyec@univ-rennes1.fr (Received 14 June 2007, revised 25 July 2007, accepted 26 July 2007) doi:10.1111/j.1742-4658.2007.06017.x Hepatitis C virus is a human pathogen responsible for liver diseases including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma Its high prevalence, the absence of a prophylactic vaccine and the poor efficiency of current therapies are huge medical problems Since the discovery of the hepatitis C virus, our knowledge of its biology has been largely punctuated by the development of original models of research At the end of the 1980s, the chimpanzee model led to cloning of the viral genome and the definition of infectious molecular clones In 1999, a breakthrough was achieved with the development of a robust in vitro replication model named ‘replicon’ This system allowed intensive research into replication mechanisms and drug discovery Later, in 2003, pseudotyped retroviruses harbouring surface proteins of hepatitis C virus were produced to specifically investigate the viral entry process It was only in 2005 that infectious viruses were produced in vitro, enabling intensive investigations into the entire life cycle of the hepatitis C virus This review describes the different in vitro models developed to study hepatitis C virus, their contribution to current knowledge of the virus biology and their future research applications Introduction The hepatitis C virus (HCV) belongs to the Flaviviridae family and is the only member of the Hepacivirus genus It is a small virus with a diameter of  50 nm, enveloped within a cell-derived lipid membrane that carries viral surface glycoproteins This envelope surrounds a capsid containing positive ssRNA The viral genome of  9600 nucleotides contains two UTR at the 5¢- and 3¢-termini and a major ORF that encodes a unique polyprotein of  3000 amino acids (Fig 1) Translation is initiated by the internal ribosome entry site (IRES) located in the 5¢-UTR Translated polyprotein is then co- and post- translationally cleaved into 10 different products: three structural proteins (the core protein and the E1 and E2 envelope glycoproteins) and seven nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) The specific enzymatic functions that have been attributed to NS2 ⁄ 3, NS3 and NS5B are serine protease, helicase and RNA-dependent polymerase, respectively HCV is a human pathogen and  170 million people are chronically infected worldwide [1] HCV infection causes major health problems because it is a principle cause of chronic liver diseases, including cirrhosis and hepatocellular carcinoma The natural history of HCV begins with a frequently asymptomatic Abbreviations HCV, hepatitis C virus; HCVcc, cellular clone of HCV; HCVpp, pseudo-particles of HCV; IFN, interferon; IRES, internal ribosome entry site; JFH1, Japanese fulminant hepatitis 1; LDLR, low-density lipoprotein receptor; NS, nonstructural; SR-B1, scavenger receptor class B type 1; VSV, vesicular stomatitis virus FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS 4705 ´ M Regeard et al In vitro HCV infection models HCV RNA (9600nt) ’UTR 3’U TR nonstructural structural Translation And Maturation F C E1 E2 NS2 NS3 p7 NS4B NS5A NS5B NS4A Glycoproteins Serine protease RNA helicase Genome encapsidation Ion channel Cysteine protease Phosphoprotein RNA-dependent RNA polymerase Membrane alterations Serine protease cofactors Fig Genetic organization and procession of HCV polyprotein A schematic representation of HCV genome is given at the top The HCV genome is composed of ssRNA encoding a large ORF flanked by 5¢- and 3¢-UTR Translation of the polyprotein precursor is mediated by the IRES contained in the 5¢-UTR The polyprotein is co- and post-translationally processed in 10 proteins by signal peptide peptidase (black solid arrows), by NS2 ⁄ autoprotease (black and large arrow) and by NS3 ⁄ 4A protease (black doted arrows) The F protein is generated by translation of an alternative reading frame but no functions have yet been attributed to this protein acute phase of infection that leads to chronic infection in  70–80% of cases Thereafter, 10–20% of chronically infected patients develop liver cirrhosis within 20 years and hepatocellular carcinoma after another decade No vaccine against HCV infection is available, and current antiviral therapies consisting of pegylated interferon (IFN) and ribavirin injections are characterized by limited efficacy, substantial side effects and high cost These clinical complications clearly document the need for more effective therapies that depend on a detailed understanding of HCV biology using appropriate experimental systems Unfortunately, research on HCV has largely been slowed by the difficulties encountered in developing efficient experimental models This review focuses on the different in vitro models of HCV that have been developed and their contribution to our current knowledge of the virus life cycle Around 10 years after discovery of the HCV genome and after many attempts to infect chimpanzees with transcripts from cloned isolates, consensus sequences of genotypes 1a, 1b and 2a were constructed These were the first viral functional sequences able to infect chimpanzees Soon after, efforts were concentrated on establishing cell culture models that support HCV replication by transfecting cells with cloned viral DNA or their derived viral transcripts Although this approach is classic in virology, it proved to be unproductive for HCV because of the very low level of replication and the high amount of input RNA needed for transfection These first studies precluded the difficulties of studying HCV in vitro 4706 Replicon system An important breakthrough was the development of cell-culture systems based on the selection of cells that support stable replication of subgenomic HCV RNAs Lohmann et al [2] worked on an HCV consensus genome of genotype 1b derived from a chronically infected patient Researchers replaced the region that encodes the core to p7 with the coding sequence of the neomycin-resistance gene and the heterologous IRES of the encephalomyocarditis virus The resulting replicon was bicistronic with translation of the first cistron (neomycin-resistance gene) being directed by the HCV IRES and that of the second cistron (NS2–5B) by the encephalomyocarditis virus IRES Other constructions were composed of a smaller second cistron encoding NS3 to NS5B proteins (Fig 2A) After transfection of Huh7 cells with this replicon, selection of the very few cells supporting autonomous replication was achieved by neomycin sulfate treatment (Fig 2Ba) Viral replication was sufficient to detect viral RNA by northern blot analysis Improvement of the system was obtained after the discovery of cell-culture-adaptative mutations that enhanced the replication efficiency by up to 10 000 times [3] These mutations are at the N-terminus of the NS3 helicase, in two distinct positions of NS4B, in the centre of NS5A and in the C-terminal region of NS5B [3–6] The significance of these mutations has been questioned because they have not been observed in wild-type viruses Moreover, insertion of some of these mutations into an infectious HCV clone reduced or completely abolished its in vivo infectivity FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M Regeard et al In vitro HCV infection models A H C V ’ UT R NS4 A H CV ’ UT R H C V ’U T R Genomic replicon B NS5 B NS4 B H CV ’ U T R ECMV IRES Neo Fig Schematic representation of the replicon system Subgenomic and genomic replicons are composed of the HCV 5¢-UTR, the gene coding neomycin phosphotransferase (NeoR), the encephalomyocarditis virus IRES, the region encoding HCV proteins and the 3¢-UTR (A) Huh7 cells are electroporated with replicon RNA Cell colonies efficiently replicating the HCV replicon are selected because of their resistance to G418 (Ba) In parallel, Huh7 subclones highly permissive to HCV replication can be obtained by G418 treatment of cells transfected with HCV subgenomic replicon Cells are then treated by IFN to eliminate the HCV replicon (Bb) NS5 A NS2 NS3 Neo Subgenomic replicon H C V ’ U TR ECMV IRES N S5 A NS3 NS4 A N S5 B NS4 B H CV ’U T R ECMV IRE S Neo C E1 E2 a Subgenomic or genomic replicon Electroporation of Huh7 cells with NS2 NS3 p7 NS4 A N S5 A NS5 B NS4 B b Subgenomic replicon G418 selection and clonal expansion Huh7 cells replicating HCV in chimpanzees [7] In parallel, the replicon system has allowed the selection of highly permissive cell clones (Fig 2Bb) Indeed, the subpopulation of Huh7 cells that supports a high viral replication rate has been cured from the replicon by long-lasting IFN treatment Two such cell lines have been generated and named Huh7-Lunet and Huh7.5 Another subclone, Huh7.5.1, has been generated similarly by curing Huh7.5 cells of replicating HCV All of these cell lines were shown to support RNA replication to a much greater extent than the parental cell line [8–10] The efficient replication of HCV in these cells may be explained by partial impairment of their antiviral defence system Indeed, Landford et al [11] suggested that some steps in the signalling pathway for detecting dsRNA were defective in the parental Huh7 cells Moreover, permissiveness of HCV replication in Huh7.5 cells is probably reinforced by the presence of a defective mutation in the RIG-I gene, which disturbs the antiviral immune response [12] The antiviral effect of IFNa observed in vivo was nevertheless reproduced in this system [4,11] Thereafter, improvements to this model were achieved with the efficient insertion of a reporter gene into the viral genome facilitating measurement of the replication activity of replicons [5,13–15] Replicons of other genotypes (1a and 2a) have also been developed IFN treatment Huh7 cells subclones [16,17] Genomic HCV replicons have also been generated and have enabled the selection of cells with stable expression of the entire viral polyprotein (Fig 2A) However, replication efficiency was lower than that observed with subgenomic replicons and no virus production was observed in these cells [16,18,19] This defect could be due to the presence of adaptative mutations that are detrimental to viral particle assembly and secretion or to the lack of some critical HCV partners in Huh7 cells Some data argue for the former hypothesis On the one hand, inoculation of chimpanzees with a Con1 sequence containing these adaptative mutations failed to establish a productive infection [7] On the other hand, production of infectious particles in Huh7 cells has been achieved with a clone named Japanese fulminant hepatitis (JFH1) constituting the so-called model of HCV cellular clone (see below) [20] Owing to its efficient replication rate, this in vitro replicon model enabled investigation into the replication process, the replication complex and host–virus interactions [21,22] Its exploitation has also enabled high-throughput screening of anti-HCV drugs targeting the replication of various genotype replicons [23] Although practical, only the replication step containing in vitro adaptative mutations can be studied with this system using viral genomes containing in vitro adaptive FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS 4707 ´ M Regeard et al In vitro HCV infection models mutations Furthermore, it should be kept in mind that, in this system, hundreds of RNA copies per cell are present, in contrast to 5–50 copies in infected hepatocytes Therefore, results should be ascertained using systems closer to HCV physiology Infection of primary cell cultures and cell lines Parallel to the development of this replication system, intensive research has aimed to discover HCV infection systems Until recently, sera obtained from infected patients or chimpanzees were the only source of HCV infectious particles However, purification of natural HCV particles from patient sera proved difficult because of the heterogeneity of their densities Viral RNA is detected in fractions ranging from 1.03 to 1.25 gỈmL)1 in a sucrose gradient Low-density HCV particles are associated with either low- or verylow-density lipoproteins [24–28] and have been shown by assays of chimpanzee model to be the most infectious fraction [29,30] HCV particles of higher densities correspond to particles associated with immunoglobulin, free particles [28,30,31] or free nucleocapsids [32] Because hepatocytes are the main target of the virus in vivo, several groups have attempted to infect primary hepatocytes or hepatic cells in vitro Thus, adult or fetal primary human hepatocytes have been shown to support HCV infection and replication in vitro [33– 36] Similarly, infections have also been conducted on primary cells obtained from other mammals, including chimpanzees and tree shrews [37,38] In these models, the replication rate was low, between 0.01 and 0.1 RNA copies per cell, depending on the experiment Therefore, highly sensitive assays were required to detect viral RNA within infected cells or in cell-culture supernatants In order to demonstrate that infection had taken place, researchers put forward supplementary data: detection of HCV negative-strand RNA, which only appears during ongoing replication; the sensitivity of replication to IFNa treatment; secretion of neosynthesized virions able to infect naive cells; and selection of quasispecies during the culture of infected hepatocytes To succeed in obtaining HCV infection in primary human hepatocytes, an existing model of hepatitis B virus infection was used to determine optimal infection conditions [39,40] Rumin et al pointed out the need to reach high levels of cellular differentiation, which they suggested may account for the ability of these cells to support virus assembly and secretion [36] Similarly, primary hepatocytes have recently been cultivated in spheroid formation because this culture con4708 dition maintains the differentiation state of the cell However, no real improvement in viral replication efficiency was achieved with this new model [41] It has also been noted that, for unknown reasons, sera from patients are not always infectious and no obvious correlation can be drawn between infectivity and viral RNA titre or with the presence of antibodies directed against structural proteins [36] Although primary human hepatocytes infected with patient sera are the most physiological in vitro model at present, the difficulty of obtaining cells and intrinsic technical constraints make it hard to use in everyday experiments This may explain the limited number of studies based on this model However, recent work using this model supported the involvement of the low-density lipoprotein receptor (LDLR) in the entry process of HCV The soluble form of LDLR, natural LDLR ligands and antibodies directed against LDLR efficiently competed with HCV infection, suggesting that LDLR is probably involved in the entry process of native HCV [42] In parallel, some groups have focused their efforts on developing an in vitro model based on hepatic cell lines [43] Among those tested, HepG2, Huh7 and PH5CH, the latter was the most susceptible to infection and replication However, the replication rate in PH5CH remained low as viral RNA could only be detected using RT-nested PCR Recently, Aly et al immortalized primary human hepatocytes with human papilloma virus E6E7 genes [44] These HPV18 ⁄ E6E7-immortalized hepatocytes could maintain hepatic-specific markers in long-term culture and were susceptible to HCV infection, as assayed by RT-QPCR detection of the intracellular HCV positive RNA strand However, viral replication efficiency was low compared with the infection system based on primary human hepatocytes (102 and 104 copies of viral RNA per microgram of cellular RNA, respectively) [42] mAbs directed against CD81, another probable component of the receptor complex, and IFNa treatment, inhibited infection and replication, respectively Interestingly, an interferon regulatory factor-7-defective form of this cell line has been engineered by stable expression of transdominant mutant interferon regulatory factor-7 These deficient cells were more susceptible to infection Indeed, hundred more copies of HCV RNA could be detected inside these cells following infection, whatever the genotype (1b, and 3) However no production of progeny viruses was shown in this infection model In some patients infected with HCV, analysis of HCV negative-strand RNA by RT-PCR indicated its presence in both the liver and haematopoietic cells [45] Growing evidence supports the idea that HCV is FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M Regeard et al also lymphotropic and lymphocytes may be an HCV reservoir In fact, Cribier et al reported the in vitro infection of primary peripheral blood mononuclear cells with high-titre sera Despite the low replication efficiency, HCV RNA was detected for a month in cell culture [46] Several other laboratories have shown that HCV could infect a B-cell line (Daudi) and T-cell lines (MT-2 and MOLT-4) in vitro [21] In a more recent study by Sung et al., proof of replication in this cell type was demonstrated by the establishment of a B-cell lymphoma cell line derived from an HCV-infected patient with type II-mixed cryoglobulinaemia [47] This cell line persistently replicated the HCV genome and produced virions that were infectious in primary human hepatocytes and lymphocytes in vitro In summary, use of HCV-containing sera to reconstitute the entire life cycle of HCV in vitro has proved to be very difficult Although infection of primary cells has been shown with convincing data, low replication efficiency and inherent technical difficulties have limited their use The development of the newly described HPV18 ⁄ E6E7-immortalized hepatocytes might constitute an easier model to conduct further analyses Parallel to the intensive research discussed thus far, other groups have developed surrogate models to investigate specific steps of HCV life cycle HCV-like particles HCV-like particles are generated by self-assembly of the HCV structural proteins and are nonreplicative The first HCV-like particle model was described by Baumert and collaborators in 1998, with particles produced in insect cells using a recombinant baculovirus containing the cDNA of HCV structural proteins of genotype 1b or 1a [48] HCV-like particles were observed by electron microscopy in intracellular compartments but were not secreted in the supernatant Consequently, purification of HCV-like particles was achieved by cell lysis followed by sucrose-gradient purification HCV-like particles are described as being 40–60 nm in diameter and of a rather high density ( 1.17 gỈmL)1) that should correspond to the density of the free viral particles contained in the serum of infected patients [48] Structural characterization of HCV-like particle envelope proteins has been conducted by analysing their antigenic properties This was done using a large panel of monoclonal and conformational antibodies directed against E1 and E2, and sera from infected patients [49,50] Results suggested that E1 and E2 located at the surface of HCV-like particles formed E1E2 heterodimers in a virion-like conformation In vitro HCV infection models This model has essentially been used in two major fields of research: binding process and vaccination development Specific binding of HCV-like particles was obtained for various hepatic and lymphocyte cell lines and also for dendritic cells, independently of CD81 expression [51,52] The limited involvement of CD81 in the binding process was further illustrated by the poor binding inhibition achieved in the presence of mAbs directed against CD81 In contrast, heparin sulfate seemed to mediate this interaction [53] Taking advantage of this binding model, a recent study showed that interaction of envelope glycoproteins with the surface of HepG2 cells induces gene expression modulations This suggested that HCV binding might induce changes in the cell that could favour HCV infection [54] In parallel, virus-like particles could constitute attractive vaccine candidates for papillomaviruses and retroviruses because they could mimic some properties of native viruses Concerning HCV-like particles, it has been shown that they are able to induce humoral and cellular immune responses in BALB ⁄ c mice, baboons and chimpanzees [55–57] Immunized chimpanzees were thereafter inoculated with HCV of homologous genotype Although vaccinated chimpanzees became infected by HCV, the infection was controlled quickly compared with unvaccinated animals [55] Although the structural, biophysical and antigenic properties of HCV-like particles have been characterized and might partly mimic those of native HCV particles and be close to pseudotyped particles described later, the binding of HCV-like particle does not require CD81 participation However, CD81 at the surface of Huh7 cells has been shown to be a crucial receptor involved in the infection process of pseudotyped viruses and cellular clones of HCV (see below) Moreover, this first HCV-like particle model does not permit investigation into HCV morphogenesis because viral budding was not observed in insect cells By contrast, Blanchard et al developed another HCV-like particle model that could be used to investigate this issue HCV-like particles were produced in mammalian cells (BHK-21) by expression of HCV structural proteins in a Semliki forest virus vector [58] Budding of HCV-like particles with a diameter of 50 nm was observed using electron microscopy and occurred at the endoplasmic reticulum membrane towards the lumen Although most of these HCV-like particles seemed to display an abortive budding process, it was shown that the correctly processed HCV core protein drives this event [59,60] However, due to the absence of complete budding, this model cannot be used to study the following steps of HCV assembly in eukaryotic cells FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS 4709 ´ M Regeard et al In vitro HCV infection models HCV pseudo particles 4710 b Reporter a CMV LTR c Gag-pol CMV HCV-E1E2 Transfection HEK 293T HCVpp production r rte r po rte Re po Re r rte po Re A few years after the development of HCV-like particles, another model was created to specifically investigate the entry process of HCV This system is called pseudo particles of HCV (HCVpp), as envelope glycoproteins of HCV are incorporated at the surface of other enveloped viruses substituting their natural envelope proteins The first constructed pseudotyped viruses were vesicular stomatitis virus (VSV) ⁄ HCV pseudotypes expressing HCV E1 and ⁄ or HCV E2 chimeric proteins These contain the transmembrane and cytoplasmic domains of envelope protein G of VSV [61] Although pseudotyped virus infectivity was neutralized by antibodies directed against E1 and E2 or by sera from HCV-infected chimpanzees or humans, these HCVpp exhibited a surprising tropism with limited infectivity on primary human hepatocytes, and better infectivity on kidney cell lines of human or nonhuman origin [61–63] One may speculate that the broad tropism of these HCVpp might be influenced by the background infectivity of VSV Moreover, some of these viruses harboured only one of the two glycoproteins suggesting that E1 and E2 could independently carry out the entry process of these HCVpp Because E1 and E2 are thought to be assembled in heterodimers at the surface of native viruses, it seems likely that both E1 and E2 are required for the infection process A second generation of HCVpp (Fig 3) has been developed using unmodified E1 and E2 envelope glycoproteins, which are exposed at the surface of retroviral particles carrying a genome with a marker gene Retrovirus budding occurs at the plasma membrane, although some data indicate that it might also exist intracellularly [64] Despite the specific endoplasmic reticulum retention of HCV envelope glycoproteins [65,66], it has been shown that in overexpression systems, a small fraction of envelope glycoproteins could be secreted to the surface membrane via the secretory pathway This led to Golgi-specific modification of glycosylation in envelope proteins [67,68] HCV envelope glycoproteins at the surface of retroviral particles are comprised mainly of correctly folded E1 and E2 assembled as heterodimers and a small fraction of E1 and E2 covalently linked in aggregates [68] In this model, expression of both E1 and E2 should derive from a unique expression construct for optimal infectivity [67,69] Various HCVpp have been developed with envelope proteins of genotypes 1a, 1b, 2a, 3a, 4a, 5a and 6a allowing analysis of cross- and genotypespecific neutralization [67,70] The presence of a marker gene packaged in these HCVpp has enabled easy Ψ CMV LTR Infection of hepatoma cell lines and analysis of reporter protein expression Fig Production of HCV pseudoparticles To produce recombinant retroviruses 293T are transfected with three expression vectors The first (a) is the packaging construct that encodes for retroviral Gag and Pol proteins After translation of the second vector (b), the RNA produced and which contains the sequence of a reporter gene could be encapsidated in particles via the presence of the retroviral encapsidation sequence (Y) The third vector (c) encodes HCV E1 and E2 glycoproteins Recombinant viruses collected from the supernatant are made up of a retroviral capsid containing a RNA genome with the HCV glycoproteins at their surface Their specific infectivity on hepatoma cell lines was analysed by expression of the reporter gene evaluation of infectivity mediated by HCV glycoproteins Using this system, the tropism of HCVpp has been studied and is, with few exceptions, liver specific [67,69–71] Because of this model, numerous questions regarding the HCV entry process could be assessed On the one hand, the receptor candidates scavenger receptor class B type (SR-B1) and CD81 have been evaluated in Huh7 cells Whereas CD81 has been shown to be directly involved in the entry process, SR-B1 influenced HCVpp entry via its cholesterol-uptake activity [68–74] In fact, high-density lipoprotein was shown to promote HCV entry and reduce the inhibitory effect of HCV-neutralizing antibodies from infected patients [73,75] On the other hand, the route of HCVpp entry has been assessed in Huh7 cells using specific drugs that increase endosomal pH or disrupt clathrin vesicles, with transdominant mutants of the GTPases Rab5 and Rab7 and small interfering RNA directed against the clathrin heavy chain These results support the hypothesis that HCVpp penetrate cells using FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M Regeard et al clathrin vesicles and passage in the early endosome is necessary for fusion between the viral envelope and an intracellular membrane [69,71,76,77] This fusion should be carried out by a fusion peptide present in viral envelope protein(s) Recently, data obtained with HCVpp suggested that E1 and E2 might both contain membrane fusion determinants, underlying a potential difference with the fusion process of other flaviviruses [78,79] Although this model has technical advantages (easy culture system and read out) and has enabled large advances in our knowledge of HCV, it represents only one category of HCV form derived from the sera of infected patients: free viruses not associated with either lipoproteins or immunoglobulins Moreover, the chimeric nature of the viruses and the intrinsic characteristics of the Huh7 cell line (its nonpermissiveness to infection with HCV-containing sera) limit absolute extrapolation of the results to native HCV Thus results should be reproduced in a cell-culture system closer to physiological conditions In vitro HCV infection models HCV RNA JFH1 5’U TR C E1 E2 NS2 NS3 p7 NS4 A 3’UTR NS5 A NS5 B NS4 B Electroporation in Huh7 cell lines HCVcc production Infection of Huh7 cell lines Expression analysis of viral protein or reporter protein Cellular clone of HCV Fig The HCVcc model Huh7 cell lines are electroporated with the RNA transcripts of the JFH1 genome A few days after transfection, viruses are secreted in the supernatant of cells replicating HCV JFH1 genome Their specific infectivity and replicative potential can be assessed on Huh7 cell lines and analysed by the expression of viral or reporter proteins or the quantification of intracellular viral RNA A major breakthrough has been achieved in the in vitro modelling of HCV propagation with the development of a cellular clone of HCV (HCVcc) This system is based on the utilization of a very particular HCV molecular clone of genotype 2a obtained from a Japanese patient with fulminant hepatitis (JFH1) First results with this JFH1 clone were obtained using the subgenomic replicon model After transfection of Huh7 cells with this replicon RNA,  4.5 · 104 cflg)1 RNA were counted, whereas only · 102 cflg)1 RNA were obtained with transfection of the con1 replicon of genotype 1b harbouring in vitro adaptative mutations Moreover, no adaptative mutations seemed to be required in the subgenomic JFH1 replicon for its efficient replication in cell culture [17] The JFH1 subgenome was also shown to replicate efficiently in other human cell lines of hepatic or nonhepatic origin (HepG2, IMY-N9, HeLa, HEK 293) and in mouse embryonic fibroblasts [80–82] Soon after the discovery of the extraordinary replication potential of the subgenomic JFH1 replicon, Wakita et al [20] described how transfection of Huh7 cells with the whole JFH1 RNA sequence led to the production of viruses shown to be infectious in vitro on naive Huh7 cells and in vivo in chimpanzees (Fig 4) Use of Huh7.5 or Huh7.5.1 cell lines further optimized the kinetics of replication and HCVcc secretion [10,83] Because of this model, it has been possible to observe cell-derived HCV particles, which showed a diameter of  55 nm when analy- sed by electron microscopy [20] In parallel, density analyses of infected-cell supernatant suggested that HCVcc are associated with lipoproteins with an heterogeneous density peak correlated to specific infectivity ranging from 1.05 to 1.1 gỈmL)1 in sucrose gradients [10,83,84] However, in vitro association of HCVcc with lipoproteins might not be as optimal as in vivo passage of this virus in chimpanzees or in mice containing human liver xenografts In fact, enhanced virus infectivity is observed after in vivo passage and is correlated with a lower density of HCV particles [85] Further analyses are needed to characterize the association between lipoprotein and HCVcc in cell culture and determine whether it reflects that seen in patient sera Most importantly, the efficiency of this model is entirely attributed to this specific JFH1 molecular clone Attempts to reproduce this model with other HCV molecular clones of various genotypes showed rather limited results with very low virus release despite demonstration of their infectivity in chimpanzees [86–88] To expand the studies to other genotypes, intergenotypic and intragenotypic chimeras have been constructed replacing the structural proteins of the JFH1 clone with those of Con1, H77, J6 and HCV452 molecular clones of genotypes 1b, 1a, 2a and 3a, respectively [89,90] With the exception of the HCV452–JFH1 chimera, efficient virus production was obtained Crossover before or inside the NS2 sequence FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS 4711 ´ M Regeard et al In vitro HCV infection models had no significant effect on replication efficiency but was shown to be critical for efficient virus production [89,90] Concerning the H77–JFH1 chimera, mutations affecting E1, p7, NS2 and ⁄ or NS3 have been detected and have contributed to improved assembly and release of viral particles [90] In addition to the utility of these chimeras to decipher the HCV entry process and assess antibody-neutralization efficiency, these experiments point to the involvement of p7 and NS2 in HCV morphogenesis and release This recent in vitro model reproducing the entire life cycle of HCV in Huh7 cell lines has enabled extensive research into various areas of HCV biology In the field of HCV entry, much effort has been employed to confirm results obtained using the HCVpp model For example, HCV entry into target cells is mediated by E2 [10], and CD81 has been shown to be critical for HCVcc infectivity In fact, antibodies directed against CD81 and CD81 downregulation with RNA interference inhibited HCVcc entry into Huh7 cell lines [10,20,83,91] Moreover, recent studies analysing the differential permissiveness of hepatic cell lines have shown that CD81 surface expression was a key requirement for HCVcc infection [83,92] SR-B1 is another putative HCV receptor that has been extensively studied with HCVpp mAbs directed against SRB1 and oxidized low-density lipoproteins, a ligand of high affinity for SR-B1, competed with HCVcc entry, supporting the hypothesis that SR-B1 is involved in HCV entry [93,94] Most recently, Claudin-1, a component of the tight junctions, has been proposed to be involved in the entry process of HCVpp and HCVcc [95] In fact, exogenous complementation of human cells expressing CD81 and SR-B1 (293T and SW13) with Claudin-1 permitted their infection with HCVpp or HCVcc Events late in the entry process of HCV have also been studied and various results supported the hypothesis drawn up using the HCVpp model: HCV uses a clathrin-dependant pathway to enter Huh7 cell lines in a pH-dependant fashion [76,96,97] In the field of virus morphogenesis, the HCVcc model enabled to incriminate p7 and NS2 in the assembly of infectious viral particles [89,90,98] Using this model, the association of lipoproteins with virions during their egress has also been suggested [84,99] However, only limited investigation could be conducted into HCV morphogenesis due to the very rare observation of particles in producing cells [100] Finally, this model has been also used to study virus–host interactions As a consequence of long-term HCVcc propagation, resistant cells are selected and adaptated viruses emerge, displaying a better infectivity partly related to a single mutation in E2 [101] 4712 Although the HCVcc model is dependent on the specific sequence of JFH1 5¢-UTR, NS proteins and 3¢-UTR, its utilization has led to great advances in our knowledge of HCV biology Urgent information is needed to determine the genetic specificity of this JFH1 molecular clone which confers high replication efficiency and virus release Nevertheless, viruses expressing exogenous marker are becoming useful tools to investigate both HCV biology and the potency of antiviral drugs [20,96,97,102] One example of the contribution of these constructs is the discovery of a protective effect that cells already replicating HCV possess against HCV superinfection [102,103] Concluding remarks Table presents all the in vitro HCV models, their potential use and limitations Increasing data about viral life cycle mechanisms have been accumulated in recent years, particularly regarding entry and replication processes The recent HCVcc model has emerged as the most useful research tool to date Several membrane receptors are involved in the HCV entry process: CD81, SR-B1, LDLR and Claudin-1 The multiplication of candidate receptors and the potential synergistic role of CD81 and SR-B1 support the hypothesis of a multistep entry pathway that may involve different receptors Moreover, one should keep in mind that HCV particles exist in patient sera in various forms, either free or associated with lipoproteins or immunoglobulin One can therefore speculate that, depending on its form, HCV may take advantage of different sets of receptors to enter into target cells Whereas the implications of CD81, SR-B1 and Claudin-1 have been ascertained using the HCVpp and the HCVcc model, the role of LDLR has been assessed in primary human hepatocytes with serum-derived HCV particles Further analyses are needed to define for each form of HCV particle, the exact sequence of events and the precise implications of these candidates as attachment or as entry receptors In parallel, growing evidence supports an internalization of HCV by endocytosis followed by passage in the early endosome However, neither the exact location of the fusion peptide in the viral surface glycoproteins nor the fusion process has been clearly documented Given the heterogeneity of serum-derived HCV particles, it cannot be excluded that multiple entry pathways may lead to productive infection with potentially different efficiencies In the field of HCV replication, a large breakthrough was the development of subgenomic replicons that allowed examination of the viral components of FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ M Regeard et al In vitro HCV infection models Table HCV in vitro models: their possible applications and limitations Reproduced viral steps Area of research Limitations Subgenomic replicon Replication in vitro adaptative mutations restricted to Huh7 cell lines artificially high level of replication Serum-derived HCV Entire life cycle HCV-LP Infection, morphogenesis HCVpp Infection replication mechanisms intracellular host defences evasion mechanisms antiviral screening entry process replication mechanisms intracellular host defences antiviral screening cell attachment vaccination morphogenesis entry process HCVcc Entire life cycle entry process replication mechanisms intracellular host defence evasion mechanisms virus production antiviral screening the replication complex and identification of the cellular partners of HCV replication Although discovery of the adaptative mutations necessary for efficient in vitro replication enabled high-throughput studies, it also raised the as yet unsolved question of the mechanisms of replication enhancement driven by these mutations More recently, the JFH1 subgenome has been shown to replicate at high levels without any adaptative mutations Analysis has shown that this may be due to a more efficient initiation of RNA synthesis by JFH1 NS5B compared with that of the Con1 clone [104] Moreover, and despite the generation of intergenotypic chimera, the HCVcc model is based on the extraordinary replication efficiency of the unique JFH1 strain and therefore requires minimal sequences of this peculiar strain: the 5¢-UTR sequence and the C-terminus NS2 or the NS3 sequence to the 3¢-UTR More information is needed to better understand this greater replication efficiency and the determinants necessary for the production and release of infectious viruses Another limitation of both the replicon system and the HCVcc model is their requirement for Huh7 cell lines that possess an impaired innate antiviral response HCVcc has also been shown to be infectious and able to replicate efficiently in the newly described HPV18 ⁄ E6E7-immortalized hepatocytes In this experiment, interferon regulatory factor-7, a regulatory factor of IFN response, was downregulated It would therefore be interesting to evaluate HCVcc infectivity few cell lines support infection technical difficulties of primary cell culture no secretion of particles independence of CD81 for entry no budding at the ER exogenous core no association of particles with lipoproteins restricted to JFH1 NS sequence restricted to Huh7 cell lines in primary human hepatocytes that possess intact antiviral responses HCVcc infection studies would then be assessed in a more physiological context and could be compared with serum-derived HCV infection Moreover, it would be of great interest to better understand all the antiviral mechanisms developed by primary human hepatocytes that may explain their limited rate of infection Acknowledgements We thank C Gamble for her critical reading of the manuscript Our research is supported by grants from the ANRS and the Ligue Nationale contre le Cancer CL and MT are supported by fellowships provided by ` the Ministere de l’Education et de la Recherche and ´ the Region Bretagne, respectively 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genomes J Virol 81, 4591–4603 Tscherne DM, Evans MJ, von Hahn T, Jones CT, Stamataki Z, McKeating JA, Lindenbach BD & Rice CM (2007) Superinfection exclusion in cells infected with hepatitis C virus J Virol 81, 3693–3703 Binder M, Quinkert D, Bochkarova O, Klein R, Kezmic N, Bartenschlager R & Lohmann V (2007) Identification of determinants involved in initiation of hepatitis C virus RNA synthesis by using intergenotypic replicase chimeras J Virol 81, 5270–5283 FEBS Journal 274 (2007) 4705–4718 ª 2007 The Authors Journal compilation ª 2007 FEBS ... culture of infected hepatocytes To succeed in obtaining HCV infection in primary human hepatocytes, an existing model of hepatitis B virus infection was used to determine optimal infection conditions... hepatocytes and lymphocytes in vitro In summary, use of HCV-containing sera to reconstitute the entire life cycle of HCV in vitro has proved to be very difficult Although infection of primary cells... be interesting to evaluate HCVcc infectivity few cell lines support infection technical difficulties of primary cell culture no secretion of particles independence of CD81 for entry no budding